PROJECT SUMMARY/ABSTRACT The therapeutic potential of hematopoietic stem cells (HSC) could be significantly enhanced by methods to generate HSC de novo from pluripotent stem cells (PSC) or reprogrammed adult cells. Thus, there has been great interest in understanding the embryologic origin of HSC and the signal pathways that guide HSC development and self-renewal, such that the process of HSC genesis can be recapitulated in vitro, a goal which has yet to be realized. Given the heterogeneity and developmental asynchrony in hemogenic precursors during their emergence in the embryo, to achieve this it will be necessary to develop better strategies to isolate and characterize the rare precursors capable of giving rise to HSC and to understand the signals that drive their development to functional, engrafting HSC. To this end, I have established that endothelial cells (EC) derived from the aorta-gonad-mesonephros region (AGM), the niche in which the first HSC emerge, can promote the in vitro specification and self-renewal of engrafting HSC from hemogenic precursors, including recent studies at the single cell level. Furthermore, I have shown that the Notch pathway functions in both specification and self-renewal of HSC in the EC niche, and that use of immobilized Notch ligands to activate the Notch pathway, along with hematopoietic cytokines, is sufficient to induce expansion of embryonic HSC. Building upon this work, the goals of this proposal are to utilize this novel in vitro AGM-EC system to elucidate the phenotypic, molecular, and functional properties of HSC precursors as they transition to functional HSC capable of long-term, multilineage engraftment. This will require isolation and functional characterization of HSC precursors across different stages of their development at the single cell level (Aim 1), followed by single cell transcriptional analysis to determine the gene regulatory networks and molecular signals promoting their development to functional HSC (Aim 2A). In studies working toward the engineering of stromal cell-free systems for HSC generation, agonists of identified signaling pathways will be functionally tested for their capacity to support HSC development in conjunction with the known requirement for Notch signaling activation (Aim 2B). Altogether, these studies will provide novel insight into the unique properties of HSC precursors, as well at the signaling mechanisms governing their maturation to functional HSC, which will have important implications in advancing our ability to generate HSC from PSC. To accomplish these aims, I will leverage the wealth of resources available in the mentoring laboratory of Dr. Bernstein, as well as key resources available at the Fred Hutchinson Cancer Research Center, the University of Washington, and Seattle Children?s Hospital, and opportunities for co-mentorship from Dr. Shahin Rafii at the Ansary Stem Cell Institute at Weill Cornell Medical College. A key aspect of my career development plan is to also acquire new skills that will facilitate my research objectives, including techniques in single cell RNA-seq and bioinformatics analysis which will be performed in collaboration with and under the co-mentorship of Dr. Cole Trapnell in Genome Sciences at the University of Washington. This type of interdisciplinary training will provide me with the tools necessary to apply innovative technologies in unravelling the complexity of niche- stem cell interactions that instruct HSC fates. My research experience studying hematopoietic stem cell development has provided me with a strong background to facilitate success in the proposed research goals. My clinical experience in caring for children undergoing hematopoietic stem cell transplantation has cemented a strong passion to expand scientific knowledge of HSC and translate this knowledge to improve therapies for children with hematologic, immunologic, and oncologic diseases. Success in my research and career development goals outlined in this proposal will enable me to establish an independent academic career as a physician-scientist with the skills to lead a diverse research team and collaborate broadly to address essential questions in stem cell biology. Ultimately, my long-term goal is to develop a laboratory-based career with focus on taking fundamental principles discovered through basic research in developmental HSC biology toward clinical applications in hematopoietic stem cell therapeutics.